Enhanced therapeutic usage of a purine nucleoside phosphorylase or nuceloside hydrolase prodrug

ABSTRACT

The use of a purine nucleoside phosphorylase or nucleoside hydrolase or a vector encoding expression of one of these enzymes is detailed along with the use of a prodrug cleaved by the purine nucleoside phosphorylase or nucleoside hydrolase for the preparation of a direct injection inhibition of replicating or non-replicating targeted cells. The targeted cells do not normally express the introduced purine nucleoside phosphorylase or nucleoside hydrolase. The enzyme and prodrug are amenable to intermixing and injection as a single dose or as separate injection or administration to the targeted cells. The substance and prodrug efficacy are enhanced through exposure of the targeted cells to X-ray radiation. Administration of a prodrug regardless of administration route to the targeted cells is effective in combination with X-ray radiation therapy to kill or inhibit function of the targeted cells.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/100,343; filed on Aug. 10, 2018; that in turn is a divisionalapplication of U.S. application Ser. No. 14/000,367, filed on Aug. 19,2013, now U.S. Pat. No. 10,080,784 B2, which is a 371 National Phase ofPCT/US/2012/025816, filed Feb. 20, 2012, which claims priority benefitof Provisional Application Ser. No. 61/444,261, filed 18 Feb. 2011, thecontents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates in general to inducing cell death throughcontrolled situ in vivo cleavage of a prodrug to yield a cytotoxic andin particular to enhancement therapeutic effect by practicing cleavagein concert with direct in situ injection of the prodrug, therapeuticradiation, or a combination thereof.

BACKGROUND OF THE INVENTION

Chemotherapy is a mainstay for treatment of many human tumors, but invivo efficacy against quiescent or slowly dividing cancers is poor(Takimoto et al. Cancer Management Handbook (11^(th) Edition), UBMMedica 2009; Sang et al. Trends Mol Med 2010; 16(1):17-26; Mellor et al.Br J Cancer 2005; 93(3):302-9). Radiotherapy and systemicallyadministered chemotherapy achieve specificity by disrupting DNAreplication, but cannot ablate quiescent tumor tissues that cycleintermittently. The inability to destroy nondividing tumor cells(including a putative tumor stem cell compartment) is acknowledged asone reason for failure against common human malignancies, includinglow-growth fraction tumors of prostate, breast, lung, and colon, amongmany others (Vessella et al. Cancer Biol Ther 2007; 6(9):1496-504;Kusumbe et al. Cancer Res 2009; 69(24):9245-53). Even in the case of asolid tumor with an uncharacteristically high mitotic index (e.g. growthfraction ˜40%), and assuming that all dividing cancer cells arecompletely destroyed by a cumulative exposure to conventional chemo- orradiotherapy, the mass would still be less than one doubling away fromachieving pretreatment dimensions.

Non-metastatic cancers of breast, prostate, larynx, and brain arecommonly treated with preoperative radiation therapy (XRT) as adebulking measure prior to definitive surgical resection (DeVita et al.Principles and Practice of Oncology (8^(th) Edition). Ronald A. DePinhoand Robert A. Weinbert, Eds. Lippincott Williams & Wilkins, 2008). Otherlocally invasive, non-metastatic tumors are suitable for life-prolongingXRT, and inoperable malignancies that obstruct a viscus (e.g., stomach,larynx, colon, or airway) are routinely treated with local radiotherapyfor palliation (Washington et al. Principles and Practice of RadiationTherapy (3^(rd) Edition). Mosby, 2009). Tumors such as these invariablyexhibit a low growth fraction, and at some point become unresponsive toboth radio- and the best available chemotherapies.

Several newer modalities have been advanced in an attempt to improvetreatment of locally invasive, non-metastatic tumors, including commoncancers such as those described above. Cryogenic, magnetic, thermal, andultrasonic cell ablative technologies, for example, have all beeninvestigated with varying degrees of preclinical or early clinicalsuccess (Osada et al. Anticancer Res 2009; 29(12):5203-9; Krishnan etal. Int J Hyperthermia 2010 Sep. 21 [Epub ahead of print]; Margreiter etal. J Endourol 2010; 24(5): 745-6). Experimental gene therapies, such asGDEPT (gene directed enzyme prodrug therapy; so-called “suicide gene”strategies), have been extensively tested, but have met with limitedsuccess against locally invasive, non-metastatic tumors for at least tworeasons (G W Both. Curr Opin Mol Ther 2009; 11(4): 421-32; Altaner etal. Cancer Lett 2008; 270(2): 191-201; Dachs et al. Molecules 2009;14(11): 4517-45). First, the efficiency of tumor cell transduction islow with currently available gene transfer vectors. The small proportionof malignant cells that express an anticancer transgene is often notadequate to mount a robust bystander effect against untransduced cellsin the tumor mass. Second, GDEPT has primarily utilized the herpessimplex virus thymidine kinase (HSV-tk) gene or prokaryotic cytosinedeaminase (CD) gene to activate intratumoral chemotherapy, and thecompounds produced by these two enzymes (gancyclovir monophosphate and5-fluorouracil (FUra), respectively) are primarily active againstdividing tumor cells. Low transduction efficiency, poor bystanderactivity, and failure to kill nondividing cancer cells account for thefailure of first generation GDEPT approaches against non-metastatic,solid tumors in the clinic.

The E. coli purine nucleoside phosphorylase (PNP) gene has been shown togenerate highly potent compounds such as 2-fluoroadenine (F-Ade) or6-methylpurine (MeP) intratumorally (Ungerechts et al. Cancer Res. 2007;67: 10939-10947; Fu et al. Cancer Gene Ther. 2008; 15: 474-484; Fu W,Lan et al. Cancer Sci. 2008; 99: 1172-1179; Parker et al. Cancer GeneTherapy In press; Gadi et al. J. Pharmacol. Exp. Ther. 2003; 304:1280-1284). Purine bases such as these diffuse freely between E. coliPNP transduced and neighboring (bystander) cells via facilitateddiffusion pathways ubiquitous in all cells, and confer a pronouncedbystander killing effect (Hong et al. Cancer Res. 2004; 64: 6610-6615).The compounds act by a unique mechanism that disrupts RNA and proteinsynthesis, and are therefore active against both dividing andnondividing (quiescent) tumor cells in vivo (Parker et al. Biochem.Pharmacol. 1998; 55: 1673-1681). Ade can be generated by intracellularE. coli PNP from prodrugs such as 2-F-2′-deoxyadenosine (F-dAdo) orfludarabine phosphate (F-araAMP) (Hong et al. Cancer Res. 2004; 64:6610-6615; Parker et al. Biochem. Pharmacol. 1998; 55: 1673-1681;Martiniello-Wilks et al. Human Gene Therapy 1998; 9: 1617-1626; Mohr etal. Hepatology 2000; 31: 606-614 ; Voeks et al. Gene Therapy 2002; 9:759-768; Martiniello-Wilks et al. J. Gene Med. 2004; 6: 1343-1357;Parker et al. Cancer Gene Therapy 2003; 10: 23-29; Parker et al. HumanGene Therapy 1997; 8: 1637-1644; Martiniello-Wilks et al. J. Gene Med.2004; 6: 43-54). The latter agent, F-araAMP, is clinically approved fortreatment of chronic lymphocytic leukemia, but has no activity againstnon-lymphoid malignancies.

F-Ade is approximately 1,000 times more active as an anticancer agentthan FUra. Despite this potency, numerous laboratories have shown thatF-Ade can be used safely as part of GDEPT. Because of 1) strongintratumoral sequestration into cellular nucleic acid, 2) slow releaseinto the systemic compartment following tumor cell death, with uptake byneighboring (bystander) cancer cells in the immediate vicinity, and 3)extensive dilution (throughout the host) of any chemotherapy releasedfrom the tumor, the approach leads to safe and consistent antitumorefficacy in numerous animal models in vivo (Ungerechts et al. CancerRes. 2007; 67: 10939-10947; Fu et al. Cancer Gene Ther. 2008; 15:474-484; Parker et al. Cancer Gene Therapy 2011 June;18(6):390-8; Gadiet al. J. Pharmacol. Exp. Ther. 2003; 304: 1280-1284; Hong et al. CancerRes. 2004; 64: 6610-6615; Martiniello-Wilks et al. Human Gene Therapy1998; 9: 1617-1626; Mohr et al. Hepatology 2000; 31: 606-614; Voeks etal. Gene Therapy 2002; 9: 759-768; Martiniello-Wilks et al. J. Gene Med.2004; 6: 1343-1357; Parker et al. Cancer Gene Therapy 2003; 10: 23-29;Parker et al. Human Gene Therapy 1997; 8: 1637-1644; Martiniello-Wilkset al. J. Gene Med. 2004; 6: 43-54; Deharvengt et al. Int. J. Oncol.2007; 30: 1397-1406; Kikuchi et al. Cancer Gene Ther. 2007; 14: 279-86).Several direct comparisons between E. coli PNP and first generationstrategies (HSV-tk and CD) indicate substantial augmentation of GDEPT bya PNP based mechanism (Martiniello-Wilks et al. Human Gene Therapy 1998;9: 1617-1626; et al. Clin Cancer Res 1997; 3:2075-80; Nestler et al.Gene Therapy 1997; 4:1270-77; Puhlmann et al. Human Gene Therapy 1999;10: 649-657). The approach has recently been approved by the Food andDrug Administration for clinical testing in the United States (IND#14271, approved Mar. 19, 2010).

The prolonged intratumoral half-life of F-Ade metabolites specificallyfollowing generation by E. coli PNP (>24 hours).(Hong et al. Cancer Res.2004; 64: 6610-6615; Parker et al. Biochem. Pharmacol. 1998; 55:1673-1681; Parker et al. Cancer Gene Therapy 2003; 10: 23-29) togetherwith bystander killing of quiescent tumor cells and tumor stem cells (byablating RNA and protein synthesis), suggested the possible use of PNPas a “point and ablate” modality for concentrating potent chemotherapywithin tumor tissues.

Thus, there exists a need to provide a more effective inhibition therapyagainst in vivo target cells and in particular tumor cells. Therefurther exists a need to improve a bystander inhibit effect againsttarget cells and to maintain such effect for a prolonged period.

SUMMARY OF THE INVENTION

The use of a purine nucleoside phosphorylase or nucleoside hydrolase ora vector encoding expression of one of these enzymes is detailed alongwith the use of a prodrug cleaved by the purine nucleoside phosphorylaseor nucleoside hydrolase for the preparation of a direct injectioninhibition of replicating or non-replicating targeted cells; thetargeted cells normally do not express the introduced purine nucleosidephosphorylase or nucleoside hydrolase. The enzyme and prodrug areamenable to intermixing and injection as a single dose or as separateinjections or other administration routes to the targeted cells.Intracellular expression of the enzyme improves efficacy. The use of theinventive substances are particularly effective in the treatment oftumors as a result of a bystander effect in which cells proximal totransfected cells and intercellular fluid enzyme inhibited function ordeath contact with the drug released by prodrug cleavage. The substanceand prodrug efficacy are enhanced through exposure of the targeted cellsto X-ray radiation.

A process for inhibiting replicating or non-replicating targeted cellsis also provided that includes the delivery of a purine nucleosidephosphorylase or nucleoside hydrolase to the targeted cells. A prodrugcleaved by the purine nucleoside phosphorylase or nucleoside hydrolaseis injected directly into proximity of the targeted cells to release apurine base cytotoxic to the targeted cells so as to kill or inhibitfunction of the targeted cells. The process is particularly well suitedfor administration into a tumor. Administration of a prodrug regardlessof whether directly into proximity to the targeted cells systemically iseffective in combination with X-ray radiation therapy to kill or inhibitfunction of the targeted cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of tumor weight as a function of time showing theeffect of different schedules of fludarabine phosphate (F-araAMP) on D54tumors that express E. coli PNP in 5% of the cells;

FIG. 2A is a plot of tumor weight as a function of time showing theeffects of intratumorally injected F-araAMP into tumors that express E.coli PNP in 10% of their cells result in antitumor activity;

FIG. 2B is a plot of tumor weight as a function of time showing theeffect of intratumoral injection of F-araAMP at high concentrations onD54 tumors;

FIG. 2C is a plot of tumor weight as a function of time showing theeffect of F-araAMP injection into tumors on D54 tumor growth in which10% of the cells express E. coli PNP activity;

FIG. 2D is a plot of tumor weight as a function of time showing theeffect of F-araAMP injection into tumors on D54 tumor growth where noneof the cells express E. coli PNP activity;

FIG. 3 is a plot of tumor weight as a function of time showing thatdirect intratumoral injection of F-araAMP (once per day or twice perday) into tumors that do not express E. coli PNP has no effect on tumorgrowth;

FIG. 4 is a plot of tumor weight as a function of time showing theeffect of direct intratumoral injection of F-araAMP directly into tumorsthat express E. coli PNP in 10% of their cells results in very goodantitumor activity, with six injections being better than threeinjections;

FIG. 5A is a plot of tumor weight as a function of time showing theeffect of adenoviral vector (Ad) PNP plus F-araAMP on D54 tumors at ahigher F-araAMP dosing regime (24 mg);

FIG. 5B is a plot of tumor weight as a function of time showing theeffect of adenoviral vector (Ad) PNP plus F-araAMP on D54 tumors at alower F-araAMP dosing regime (18 mg);

FIG. 5C is a plot of tumor weight as a function of time showing theeffect under varied sequences of administration and differing origin PNPsequences at still low F-araAMP dosing (3 mg);

FIG. 6A is a plot of tumor weight as a function of time showing theefficacy of F-dAdo through direct intratumoral injection into tumors onD54 tumor growth, where none of the cells express E. coli PNP activity;

FIG. 6B is a plot of tumor weight as a function of time showing theefficacy of F-dAdo through direct intratumoral injection into tumors onD54 tumor growth in which 10% of the cells express E. coli PNP activity;

FIG. 7A is a plot of tumor weight as a function of time showing theeffect of intratumoral F-araAMP and Ad/PNP on DU145 tumors;

FIG. 7B is a plot of tumor weight as a function of time showing theeffect of intratumoral F-araAMP and Ad/PNP NCI-H322M non-small cell lungadenocarcinoma tumors;

FIG. 8A is a plot of tumor weight as a function of time showing theeffect of intratumoral injection of F-araAMP plus radiation on D54tumors in which 10% of cells express E. coli PNP;

FIG. 8B is a plot of tumor weight as a function of time showing theeffect of intratumoral injection of F-araAMP plus radiation on 10%D54/PNP tumors for varied radiation regimes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention has utility in inhibiting a target cell mass andin a specific embodiment, in vivo tumor growth. It has been surprisinglydiscovered that direct intratumoral injection of a prodrug that uponcleavage by an enzyme yields a cytotoxic base to a tumor containing orexpressing the enzyme is more efficacious than conventional routes ofdelivery including orally, parenterally (e.g., intravenously),intramuscularly, intraperitoneally, or transdermally.

Direct intratumoral injection of a prodrug has conventionally beendiscounted as unnecessary and equivalent to systemic prodrug deliveryowing to the well vascularized nature tumors. Administration of such aprodrug, regardless of administration route, to cells expressing orproximal to the enzyme is rendered more effective when coupled withtumor irradiation. This is considered surprising as PNP is not known tobe a radiation sensitizer, alone or in the presence of a prodrugsubstrate.

The present invention is based on injection of a prodrug such asfludarabine phosphate into proximity to target cells expressing non-hostpurine nucleoside phosphorylase (PNP) or nucleoside hydrolase (NH)results in high level bystander inhibition and killing; and destructionof large human tumor xenografts in murine models in vivo. Non-host PNPor NH and prodrug substate for the enzyme, regardless of the rate ofprodrug administration augments radiotherapy and works by a uniquemechanism. In particular, intratumoral generation of 2-fluoroadenine orother toxic purine from a prodrug and which is slowly released to thesystemic compartment and greatly diluted in the host has improvedeffect. Injection of large subcutaneous tumors with an adenoviral vectorexpressing E. coli PNP followed by fludarabine phosphate or otherprodrugs results in tumor regressions and prolonged inhibition of tumorgrowth. An anticancer process is provided in which tumors resistant toavailable agents are safety infiltrated by treatment repetitions (e.g.,on a daily basis) to confer 1) trapping of potent chemotherapy withincancer tissue, and 2) destruction of malignant parenchyma in atitratable fashion.

Prolonged target cell inhibition is promoted by direct injection of theprodrug to the target cells in a sustained release formulation.Prolonged release of prodrug promotes inhibition of tumors with a lowgrowth fraction.

An enzyme operative herein is a nonhuman purine nucleoside phosphorylase(PNP) or nucleoside hydrolase (NH) such as that obtained from E. coli,Trichomonas vaginalis, or indeed any other nonhuman PNP which canconvert a prodrug substrate to produce a cytotoxic purine base. Non-hostnucleoside hydrolases along with a suitable prodrug are appreciated toalso be operative herein as a basis to practice the present invention.The prodrug, through hydrolase cleavage, is selected to produce acomparatively higher cytotoxicity compound. It is further appreciatedthat mutant PNPs and hydrolases such as those detailed in U.S. Pat. No.7,488,598 are operative herein to generate a cytotoxic purine base fromthe prodrug and those cells of a human subject that have beentransfected or are simply in proximity to the enzyme. It is appreciatedthat an enzyme as used herein affords a cytotoxic purine base ofsufficient potency to generate a bystander effect thereby inhibitingtransfected cells, transduced cells, as well as bystander cells.

As used herein “proximity” is intended to mean introduction directlyinto a defined tissue mass, such as for example a tumor mass, as well asadjacent to a target cell within a spacing of at least 50 adjacent celldiameters or equivalent linear spacing.

A prodrug operative herein has the attribute of being relativelynontoxic to subject cells yet upon enzymatic cleavage of the prodrugproduces a cytotoxic purine base. Representative prodrugs are known tothe art (Ungerechts et al. Cancer Res. 2007; 67: 10939-10947; Fu et al.Cancer Gene Ther. 2008; 15: 474-484; Fu et al. Cancer Sci. 2008; 99:1172-1179; Parker et al. Cancer Gene Therapy In press; Gadi et al. J.Pharmacol. Exp. Ther. 2003; 304: 1280-1284; Hong et al. Cancer Res.2004; 64: 6610-6615; et al. Biochem. Pharmacol. 1998; 55: 1673-1681;Martiniello-Wilks et al. Human Gene Therapy 1998; 9: 1617-1626; Mohr etal. Hepatology 2000; 31: 606-614; Voeks et al. Gene Therapy 2002; 9:759-768; Martiniello-Wilks et al. J. Gene Med. 2004; 6: 1343-1357;Parker et al. Cancer Gene Therapy 2003; 10: 23-29; Parker et al. HumanGene Therapy 1997; 8: 1637-1644; Martiniello-Wilks et al. J. Gene Med.2004; 6: 43-54). While the following data details the operation of thepresent invention in the context of F-dAdo and F-araAMP, it isappreciated that these results are extendable to other prodrugs.

A D54 human glioma model is selected to investigate safety and efficacyof E. coli PNP/F-araAMP for several reasons. First, D54 tumors in miceare refractory to conventional chemotherapeutic agents, includingcompounds such as BCNU that are clinically approved for human gliomatreatment. Second, the D54 model is relatively slow growing in mice(doubling time of 10 to 15 days), and provides a means to test whetheran approach kills both dividing and nondividing tumor cells. BecauseF-araAMP administration schedules described below are given over athree-day period, complete regression or cure establishes destruction ofthe non-proliferative compartment of a tumor mass. Third, human gliomashave been a target for clinical testing of GDEPT. Human D54 tumors arehighly resistant to conventional chemo-, radio-, and gene therapy-basedinterventions. It should be noted that although a glioma model isselected for the present analysis, the established mechanism of cellkilling by purine bases such as F-Ade or other prodrugs (disruption ofRNA and protein synthesis) is active against diverse malignant celltypes. (Parker et al. Biochem. Pharmacol. 1998; 55: 1673-1681). Theexperiments below describe ablation of quiescent tumor cells followingeither lentiviral transduction or adenoviral gene delivery. It isappreciated that other tumors including DU145, as well as numerous othergene transfer vectors is and prodrug substrates for encoded enzymes areoperative herein.

To explore the breadth and efficacy of the inventive therapy, theNCI-H322M non-small cell lung adenocarcinoma cell line is also studied.Conventional chemotherapy and radiotherapy protocols afford sufferers ofthis cancer a five year survival of less than 15%. NCI-H322M shares manyof the attributes of D54 human glioma in that the tumors are generallyrefractory and slow growing with a doubling time of 30 to 35 days. Theability of the present invention to kill both dividing and non-dividingNCI-H322M cells demonstrates the generality of the claimed inventionbeing able to kill or otherwise in inhibit a variety of target cellstypes.

The present invention details a process for generating a very potentcytotoxic agent specifically within a target cell volume in general andspecifically in tumor parenchyma. The inventive strategy has been shownsafe based on the limited radius of F-Ade diffusion following generationwithin a tumor mass and extensive dilution (to unmeasurable F-Ade levelsin serum) after release from dying tumor cells and confers consistent invivo bystander killing. The inventive mechanism of antitumor activityalso differs fundamentally from all other approaches to GDEPT. Antitumoractivity of F-Ade is due to disruption of RNA and protein synthesis,which causes ablation of both dividing and non-dividing tumor cells.(Parker et al. Biochem. Pharmacol. 1998; 55: 1673-1681.) The findingthat relatively slow growing D54, NCI-H322M, or DU145 tumors (doublingtime of 7 to 15 days) are completely destroyed by three days of IP or ITtreatment (FIGS. 1, 2A-2D, 7B and 5A-5C) indicates activity against bothcycling and noncycling tumor cells in vivo.

Intratumoral injection of F-araAMP of the present invention minimizessystemic exposure to the prodrug and maximizes drug levels within tumortissue the target cell mass such as itself. Pronounced antitumoractivity following intratumoral injection of F-araAMP or other prodrugsis noted in the setting of PNP or NH expression. The combination ofAd/PNP with intratumoral F-araAMP injection is noted herein to besignificantly more efficacious than Ad/PNP followed by systemic F-araAMPaccording to the present invention. It is noted that prodrug injectionis effective when administered in a single bolus with PNP or NH, or ininjections preceding or succeeding the non-human PNP or NH enzymepresence in the parenchyma.

These results suggest a straightforward means for applying a comparablestrategy in human subjects and without the need for modification ofvector tropism, enhanced bystander killing, or retargeting. Both theadenoviral vector and F-araAMP have been comprehensively studied inprevious clinical trials. Moreover, the doses of F-araAMP given as localtherapy to treat 300 mg tumors in mice (3 to 24 mg administered 3 times)are much less than the amount of F-araAMP routinely administered as partof standard clinical care in humans (˜40 mg per dose×5 daily doses givenevery 4 weeks). The present invention provides a therapeutic modality inwhich Ad/PNP followed by F-araAMP are administered repeatedly toneedle-accessible tumors (prostate, breast, head and neck, or withradiology guidance, other tumor masses) on a frequent (e.g., daily)basis to sequentially destroy large regions of a tumor while minimizingsystemic exposure to either F-araAMP, F-Ade, or other PNP cleavedprodrug. A “point and ablate” approach is feasible specifically for thePNP GDEPT approach because of the potent antitumor activity of F-Ade andits high bystander activity, together with activity againstnonproliferating tumor cells. Intratumoral generation of F-Ade shouldprovide a means to concentrate the agent intratumorally and minimizesystemic exposure in the host.

Radiotherapy primarily targets actively dividing tumor cells, but failsto ablate quiescent tumor tissues, particularly in areas of necrosis(Puhlmann et al. Human Gene Therapy 1999; 10: 649-657). The presentinvention provides an enhancement of radiotherapy in combination withPNP or NH prodrug. Very potent anticancer agents that work through amechanism distinct from XRT are provided herein to treat the non-cyclingcompartment of solid malignancies. F-Ade, which potently kills bothdividing and nondividing tumor cells, is a preferred prodrug in thatregard. Common tumors that are administered radiation therapy prior tosurgical resection (glioma, breast, prostate, head & neck, lung, andother cancers treated with curative or palliative intent) also benefitfrom an inventive combination therapy.

In the method of the invention described above, the mammalian cells tobe killed can be tumor cells. Cells comprising any solid tumor, whethermalignant or not, can be killed by the present method based on theability to transfer or express the PNP or NH gene selectively to atleast a small percentage of cells comprising the tumor. For example, ithas been shown that intravenous injection of liposome carrying DNA canmediate targeted expression of genes in certain cell types. Targeting ofa PNP or NH gene or expression of the gene to a small fraction of thecells in a tumor mass followed by substrate administration could beadequate to mediate involution. (Zhu et al. Science 261:209-211, 1993)Through the substantial bystander effect and killing of nondividingcells demonstrated in the Examples, the present method can destroy thetumor. Although, in the exemplified method, the mammalian cells arehuman glioma, non-small cell lung adenocarcinoma, and prostate cells, itcan be appreciated that the methods taught herein can be applied toother cells and their susceptibility to the present methods can bedetermined as taught.

In addition to killing tumor cells, the method of the invention can alsokill virally infected cells. In a virus-killing embodiment, the genetransfer method selected would be chosen for its ability to target theexpression of PNP in virally infected cells. For example, virallyinfected cells may utilize special viral gene sequences to regulate andpermit gene expression (i.e., virus specific promoters). Such sequencesare not present in uninfected cells. If E. coli or other PNP genes areoriented appropriately with regard to such a viral promoter, PNP wouldonly be activated within virally infected cells, and not other,uninfected, cells. In this case, virally infected cells would be muchmore susceptible to the administration of MeP-dR or other substratesdesigned to be converted to toxic form by PNP.

In other applications of the present invention, a medicament is providedto kill or otherwise inhibit the function of any desired target cellvolume of a subject. The broad applicability of the present invention tokill or otherwise inhibit function of cells affords clinicalpractitioners with superior control of administration, as well asimproved healing profiles over a variety of conventional procedures. Thepresent invention affords a chemical cellular ablation alternative toprocedures involving cautery, excission. It has been surprisingly notedthat the chemical cellular ablation afforded by the present inventionprecludes the granulation and scarification associated with cautery orexcission techniques thereby providing a superior healed tissue aroundthe situs of chemical ablation and as a result, the present inventionhas uses in the treatment of cardiac arrhythmia, cyst, reduction,ganglion treatment, male sterilization, cosmetic dermatologicalprocedures, and melanoma treatment. It is appreciated that chemicalcellular ablation according to the present invention is readilyperformed by administration of PNP or NH enzyme, genes expressing thesame form of a viral vector as detailed herein; along with proximaldelivery of a prodrug for the PNP or NH. Based on the location of thetarget cells for chemical cellular ablation, inventive medicament isadministered via a catheter, microsyringe, canula, or syringe; as wellas topically in a cream base. Preferably, the PNP or NH enzyme isexpressed intracellularly.

An isolated nucleic acid encoding a non-human or genetically modifiedhuman purine nucleoside phosphorylase or nucleoside hydrolase in amammalian cell is provided in the present invention. More specifically,the invention provides an isolated nucleic acid encoding an E. coli PNPin a mammalian cell. By “isolated” is meant separated from other nucleicacids found in the naturally occurring organism from which the PNP geneis obtained.

As described above, in a preferred embodiment, the PNP or NH used in thepresent methods can include genetically modified human or nonhumanmammalian PNP or NH capable of reacting with a substrate that the nativePNP or NH in the cell to be killed will not recognize or recognizes verypoorly. Thus, the nucleic acids of the invention that encode the PNP orNH of the invention are present in cells in which they are not naturallyfound, either because they are from a different organism or because theyhave been modified from their natural state. The key requirement of thenucleic acids encoding the PNP or NH is that they must encode afunctional enzyme that is able to recognize and act upon a substratethat is not well recognized by the native PNP or NH of the cell.

A eukaryotic transfer vector comprising a nucleic acid encoding anon-human or genetically modified purine nucleoside phosphorylase ornucleoside hydrolase is also provided. The vector must be capable oftransducing or transfecting at least some percentage of the cellstargeted. The transfer vector can be any nucleotide construction used todeliver genes into cells (e.g., a plasmid), or as part of a generalstrategy to deliver genes, e.g., as part of recombinant retrovirus oradenovirus (Ram et al. Cancer Res. 53:83-88, 1993). The Examples providea plasmid vector containing a nucleic acid encoding PNP.

The vector of the invention can be in a host capable of expressing afunctional PNP or NH. As used in the method of the invention, the hostcell is the cell to be killed, which expresses the PNP or NH and iskilled by the toxic product of the reaction of the enzyme and theprodrug that is an enzymatic substrate. A method of determiningsusceptibility is provided in the Examples which teach protocols for thetransfection of host cells, and demonstrate the expression of PNP andtoxicity to the host cells in the presence of substrate. Alternatively,the active enzyme or pro-enzyme thereof is administered into a targetcell mass.

In addition to the present gene transfer methods, the PNP gene productcan also be selectively delivered to the tumor cells by a number ofdifferent mechanisms and this PNP could be used to produce F-Ade at thesite of the tumor.

For instance, the PNP or NH enzyme can be attached to any desiredmonoclonal antibody and injected into the patient. After allowingsufficient time for the clearance of all PNP or NH conjugated tomonoclonal antibody that has not bound to the target, the patient istreated with F-araAMP, which is cleaved to F-Ade only at the targetedsite. Such a procedure requires only the availability of an appropriatemonoclonal antibody. The procedures used for conjugating proteins totarget-specific monoclonal antibodies are routinely available. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter et al. BioconjugateChem. 2:447-451, 1991; Bagshawe et al. Br. J. Cancer 60:275-281, 1989;Bagshawe et al. Br. J. Cancer 58:700-703, 1988; Senter et al.Bioconjugate Chem. 4:3-9, 1993; Battelli et al. Cancer Immunol.Immunother. 35:421-425, 1992; Pietersz and McKenzie Immunolog. Reviews129:57-80, 1992; and Roffler et al. Biochem. Pharmacol 42:2062-2065,1991). Other ligands, in addition to monoclonal antibodies, can beselected for their specificity for a target cell and tested according tothe methods taught herein.

It is also possible to entrap proteins in liposomes and target them tospecific tissues. The PNP or NH gene product can, thus, be selectivelydelivered to a tumor mass using targeted liposomes. After allnon-targeted liposome is cleared from the blood, the patient is treatedwith F-araAMP which is cleaved to F-Ade by the PNP only at the targetedsite. Once again, this procedure requires only the availability of anappropriate targeting vehicle. The following references are examples ofthe use of this technology to target specific proteins to tumor tissue(Hughes et al. Cancer Research 49:6214-62210, 1989; and Litzinger andHuang Biochimica et Biophysica Acta 1104:179-187, 1992).

A prodrug that represents enzymatic substrate for a non-host PNP or NHis injected directly into target cell mass as for example,intratumorally in a pharmaceutically acceptable carrier such as forexample saline or alternatively is encapsulated to modify prodrugstability and/or therapeutic characteristics. An inventive prodrug isreadily administered as a gel, paste or capsulated withinmicroparticles. It is appreciated that such carriers for prodrugs arereadily used to provide a prolonged release of the prodrug, modifieddiffusion within the targeted cell mass, and storage stability ascompared to dissolution in a saline solution. With resort tomicroparticles, release rates of an inventive prodrug are readilyextended to more than one week, more than two weeks, even beyond sixweeks. (Zentner et al., J. control release 72 (1-3): 203-215, 2001). Aninventive prodrug is readily prepared and injected in a paste ofpolylactic acid, poly(epsilon-caprolactone), or a combination thereof(Jackson et al., Cancer research 60 (15): 4146-4151, 2000). Prodrugs arealso suitably encapsulated within microspheres from a variety ofmaterials including polylactic acid, poly(epsilon-caprolactone),polyvinyl pyrrolidone, hydroxypropylcellulose, methyl cellulose, andother polysaccharides (Harper et al, Clin. Canc. Res. 5:4242-4248, 1999;Dordunno et al., Cancer Chemother. Pharmacol. 36: 279-282, 1995; Bert etal., Cancer Lett. 88:73-78, 1995;) It is appreciated that with acontrolled release formulation of prodrug, larger dosings of prodrug areinjected into a target cell mass less frequently to achieve a prolongedcell inhibition and bystander effect.

Injection of the enzyme and prodrug into a target cell volume such as atumor, can be performed for monetary compensation. With the subject havean undesired growth or function of targeted cells compensating aprovider of injection according to the present invention for the effortof inhibiting function or even killing the target cells.

The present invention is further detailed with respect to the followingnonlimiting examples. These examples are not intended to limit the scopeof the appended claims.

EXAMPLES Example 1 Studies with Human Tumor Xenografts in Mice

Parental and E. coli PNP expressing D54MG (human glioma) tumor cells(2×10⁷ cells) are injected subcutaneously into the flanks of nude mice(nu/nu) purchased from Charles River Laboratories (Wilmington, Mass.).D54 tumor cells stably transduced with E. coli PNP are prepared asdescribed previously (Parker et al. Cancer Gene Therapy 2011June;18(6):390-8). Tumors are measured with calipers and an estimate ofthe weight calculated using the equation, (length×width)/2=mm³, andconverted to mg assuming unit density. Unless stated otherwise,therapeutic drugs and the adenoviral vector expressing E. coli PNP(Ad/PNP) are injected into D54 tumors in 150 μl volumes by 8 separateinjections of approximately 20 μl each in an effort to evenly distributethe administered agent. Each treatment arm of each group contained atleast 6 mice. Mice are monitored daily for weight loss and twice weeklyfor tumor dimensions. T-C (tumor growth delay) is taken as thedifference in days to 2 doublings between drug-treated andsaline-treated groups. The time to the evaluation point for each animal(2 doublings) is used as the end point in a Student's t-test, theMann-Whitney rank sum test, or a life table analysis in order tostatistically compare growth data between treatment groups. Allprocedures are performed in accordance with a protocol approved by theIACUC of Southern Research Institute. F-araAMP is obtained from ScheringA. -G. (Berlin, Germany). In this and subsequent examples, F-Ade isobtained from General Intermediates of Canada, Inc. (Edmonton, Alberta,Canada). Treatments are initiated when tumors are 250 to 300 mg (˜1-1.5%of total animal weight).

Example 2 Measurement of E. Coli PNP Activity

The proportion of lentiviral transduced cells in a tumor mass isverified by measuring E. coli PNP activity in representative cancersremoved from mice on the first day of drug treatment. Crude extracts areprepared as described previously (Parker et al. Human Gene Therapy 1997;8: 1637-1644) after tumor excision from the flanks of mice. The extractsare incubated with 50 mM PO₄, 100 μM 6-methylpurine-2′-deoxyriboxide(MeP-dR), and 100 mM HEPES buffer (pH 7.4) at a concentration of extractthat resulted in a linear reaction over the incubation period. Theformation of MeP is monitored using reverse phase HPLC. By convention,one unit of PNP activity is defined as the amount of extract necessaryto cleave 1 nmole of MeP-dR per mg protein in a 1 hour period.

Example 3 Monitoring Intratumoral Metabolism of F-araAMP

Total radioactivity is determined after injection of 3 mg [8-³H]F-araAMP(10 μCi) into 300 mg D54 flank tumors. [8-³H]F-araAMP is obtained fromMoravek Biochemicals Inc. (Brea, Calif.). Tumors are removed from themice at 10 minutes or 4 hours after injection and dissolved in 1 ml ofSoluene 350 (Packard Instrument, Meriden, Conn.) by incubating at 55° C.for 4 hours. A portion of each extract is mixed with scintillation fluidand radioactivity determined.

Example 4 Effect of Different Schedules of Fludarabine Phosphate(F-araAMP) on D54 Tumors that Express E. Coli PNP in 5% of the Cells

Parental D54 tumor cells are mixed with D54 tumor cells stablytransduced with E. coli PNP so that 5% of the mixture expressed the PNPtransgene. This 95/5 mixture is injected sc into the flanks of nudemice. Intraperitoneal treatment with F-araAMP (250 mg/kg once per dayfor 3 consecutive days; 167 mg/kg 3 times per day for 3 consecutivedays; 100 mg/kg 5 times a day for 3 consecutive days; or vehicle control5 times a day×3 days) began on day 17 when tumors are approximately 250mg. The activity of E. coli PNP in the tumors at the time of treatment(day 17) is 2,500±400 units. Tumor growth in all F-araAMP treatmentgroups is significantly different than that in vehicle treated groupP<0.001.

FIG. 1 depicts the antitumor activity of E. coli PNP plus fludarabinephosphate (F-araAMP) when 5% of tumor cells (transduced with the geneprior to implantation) express the recombinant enzyme. Intraperitoneal(systemic) administration of F-araAMP (100 mg/kg given 15 times, 167mg/kg given 9 times, or 250 mg/kg given 3 times) led to completeregressions of all tumors and cures of all mice. It has been shownpreviously that parental D54 tumors (i.e. without E. coli PNP) are notsensitive to treatment with F-araAMP and that tumor regressions withF-araAMP in this setting exhibiting dose dependence on the fraction oftumor cells expressing E. coli PNP (Hong et al. Cancer Res. 2004; 64:6610-6615; Parker et al. Human Gene Therapy 1997; 8: 1637-1644; also seebelow). Tumors in which 100% of cells are transduced with E. coli PNPand non-transduced (parental) tumors grow at similar rates (Hong et al.Cancer Res. 2004; 64: 6610-6615), suggesting that a ˜5% level oftransduction would be maintained throughout expansion of the tumormodel. To verify this assertion, tumor extracts are prepared from threerepresentative tumors taken from mice on the first day of drug treatmentand levels of E. coli PNP determined to be 2,500±400 units. E. coli PNPactivity of 126,000 units is present in tumors comprised of 100% PNPexpressing cells from this cell line (Hong et al. Cancer Res. 2004; 64:6610-6615). The findings therefore suggest that in this particularexperiment the parental (no PNP-expression) cells grew slightly morerapidly than the PNP transduced line in vivo, and confirm thattransduction percentage described in FIG. 1 is ≤5%, and perhaps closerto 2-3%.

The results shown in FIG. 1 establish that complete regressions or curesof large tumors (approximately 1% of the total body weight of theanimal) can be safely accomplished by an inventive PNP-based GDEPTstrategy. The findings also demonstrate excellent in vivo bystanderactivity. As few as three intraperitoneal (IP) injections of F-araAMPinstant (non-sustained release) led to destruction of large tumors,although ≤5% of cells expressed the activating gene. Moreover, treatmentwith F-araAMP resulted in only a 10 to 20% decrease in body weight,which is quickly regained after the F-araAMP schedule is completed.There is no gross tissue damage in the region immediately surroundingthe tumor or other evidence of undesired sequelae despite a substantialprolongation of life. A single IP, injection of 3× dose F-araAMP 30% byweight in polylactic acid microspheres achieves a similar effect.

Example 5 Intratumoral Injection of F-araAMP on D54 Tumors

In FIG. 2A, parental D54 tumor cells are mixed with D54 tumor cells thathad been transduced with E. coli PNP to a final proportion in which 10%expressed the transgene. This 90/10 mixture is injected sc into theflanks of nude mice. Tumors are injected once per day for 3 consecutivedays starting on day 17 with 150 μl of saline, 1.5 mg of F-araAMPdissolved in saline, or 3 mg of F-araAMP is dissolved in saline. Theactivity of E. coli PNP in the tumors at the time of treatment (day 17)is 14,000±2,600 units. Tumor growth in the 3 mg treatment group issignificantly different than that in the vehicle treated group (P<0.001)but is not significantly different than that in the 1.5 mg treatmentgroup (P=0.458). In FIG. 2B, parental D54 tumor cells (no E. coli PNPexpression) are injected sc into the flanks of nude mice. Tumors areinoculated once per day for 3 consecutive days starting on day 17 with150 μl l of saline, 3 mg F-araAMP is dissolved in saline, 0.15 mg ofF-Ade dissolved in saline, 1.26 mg of F-Ade dissolved in 150 mL DMSO,0.63 mg of F-Ade dissolved in DMSO, or DMSO. This experiment is repeatedwith a single injection of 9 mg F-araAMP 30% by weight in polyactic acidmicrospheres in saline with similar results. The tumor growth in micetreated with 0.63 or 1.26 mg F-Ade dissolved in DMSO is significantlydifferent than that in the DMSO vehicle treatment group (P=0.011 and0.002, respectively).

In FIG. 2A, it is shown that injection of F-araAMP into tumors in which10% of the cells express E. coli PNP has a strong antitumor effect. Notethat the dose of F-araAMP in this experiment is considerably below themaximally tolerated dose and that additional injections are made withgreater antitumor activity. In the low dose of F-araAMP, the PNPactivity in the tumors 30 days after treatment with F-araAMP is 1,200units, whereas at the time of treatment the PNP activity in the tumorsis 14,000 units. This result indicates that the F-araAMP treatmentpreferentially killed cells expressing E. coli PNP, which suggests thatthe antitumor activity is dependent on the expression of E. coli PNP.The amount of F-araAMP injected is 120 mg/kg which is much less than themaximally tolerated dose at this schedule (750 mg/kg, q1d×3). Betterresults are seen at higher doses of F-araAMP when dissolved in DMSO;FIG. 2C.

Example 6 Intratumoral Injection of F-araAMP in D54 Tumor Cells With andWithout PNP

In FIGS. 2B, 2D and 3 it is shown that direct intratumoral injection ofF-araAMP into tumors in which no cells express E. coli PNP activity hasno effect of tumor growth. This result indicates that the antitumoractivity of F-araAMP shown in FIGS. 2A, 2C and 4 is due to theexpression of E. coli PNP in some of the tumor cells. The antitumoractivity of F-Ade (the active metabolite of F-araAMP) is also injectedinto tumors in the same manner as F-araAMP. 0.15 ml of 1 mg/ml F-Adedissolved in water (limit of solubility) showed no antitumor effect.0.15 ml of 8.6 mg/ml F-Ade dissolved in DMSO is injected into tumors; amodest antitumor effect is observed.

These results indicate that a product containing both a vector (todeliver E. coli PNP) and time-released F-araAMP is effective in thetreatment of local cancers that otherwise are untreatable. At least oneinjection of this product would inhibit metabolism or even kill any cellthat expresses E. coli PNP plus many bystander cells with reducedsystemic toxicities associated with F-araAMP. This is repeated as manytimes as necessary to abolish a tumor mass. For example, a patient isinjected once per week on an outpatient basis with reduced toxicityuntil the tumor is completely eliminated. Multiple intratumoralinjections of F-araAMP resulted in greater antitumor activity (FIG. 4).

Since only a fraction of F-araAMP injected IP actually perfuses amalignant tumor in vivo, it is tested whether injecting F-araAMPdirectly into the tumor mass could enhance efficacy. An initial study isshown for tumors in which 10% of cells expressed E. coli PNP (FIG. 2A).Intratumoral injection of 3 mg of F-araAMP per injection (3 totalinjections) conferred significant antitumor effects (p<0.001 with littleor no weight loss (less than 4%), whereas F-araAMP has no effect wheninjected into parental (no E. coli PNP expressing) tumors (FIG. 2B).Because the weight of a mouse is approximately 0.025 kg, injection of 3mg F-araAMP is equivalent to a dose of 120 mg/kg F-araAMP, which is 25to 50 percent of the total systemic amounts studied in FIG. 1. Injectionof F-Ade (the active metabolite of F-araAMP) into tumors also fails toelicit antitumor activity when tested at the highest possible solubilityin saline (FIG. 2B). F-Ade is also dissolved in DMSO, and threeinjections of 1.26 mg F-Ade (the approximate molar equivalent of F-Adein a 3 mg injection of F-araAMP) into the tumor resulted in minimalantitumor activity (FIG. 2B; p=0.011 with respect to mice injected withDMSO vehicle), but caused a 10% decrease in body weight. Threeintratumoral injections of either 2.5 or 5 mg of F-Ade (dissolved inDMSO) led to death in 3 of 6 and 4 of 6 mice, respectively, whichindicated that 1.26 mg is very near its maximally tolerated dose (MTD).The results therefore demonstrate that unlike IT F-araAMP followingintratumoral expression of E. coli PNP, direct IT injection of F-Ade haslittle antitumor efficacy.

As noted above, the amount of F-araAMP given intratumorally (in FIG. 2A)is less than the total intraperitoneal dose described in FIG. 1. Inorder to investigate the impact of higher doses of F-araAMP, theF-araAMP is dissolved in DMSO, and given at concentrations welltolerated IT, but well above the maximally-tolerated dose ifadministered IP (Hong et al. Cancer Res. 2004; 64: 6610-6615). Threedoses of 6 or 24 mg is administered by IT injection to tumors in which10% of cells expressed E. coli PNP (FIG. 2C). Parental D54 tumor cellsor mixtures of D54 tumor cells in which 10% of the cells expressed E.coli PNP are injected sc into the flanks of nude mice. Tumors areinjected once per day for 3 consecutive days starting on day 17 with 150μl of DMSO, 24 mg F-araAMP dissolved in DMSO, or 6 mg F-araAMP dissolvedin DMSO. The activity of E. coli PNP in the D54 tumors in which 10% ofthe cells expressed E. coli PNP (on day 17) is 8,600±620 units. Thetumor growth in the mice bearing PNP tumors and treated with 6 or 24 mgof F-araAMP is significantly different than the vehicle treated group(P=0.014 and <0.001, respectively), but is not significantly differentfrom one another in this experiment. Three of the 6 tumors treated with24 mg of F-araAMP became ulcerated (days 17, 21, and 24), requiringsacrifice of the study animals. The remaining 3 tumors completelyregressed and mice remained tumor-free until the experiment is ended onday 70. There are no ulcerations in tumors treated with 6 mg ofF-araAMP, and a single course of this treatment led to robust tumorregressions and a prolonged antitumor effect. Injection of F-araAMP atthe highest dose resulted in a modest (7%) decrease in body weight,which recovered rapidly following completion of drug treatment.Injection of F-araAMP at these doses into parental tumors (noPNP-expression) did not result in antitumor activity.

Example 7 Effect of Intratumoral F-araAMP and Ad/PNP on D54 Tumors

Modest antitumor activity after intratumoral (IT) injection of areplication deficient adenoviral vector expressing E. coli PNP (Ad/PNP)followed by systemic treatment with F-araAMP has been demonstrated (Honget al. Cancer Res. 2004; 64: 6610-6615). Based on the high activitydemonstrated in FIG. 2C when high doses of F-araAMP are administered IT,the antitumor activity of Ad/PNP plus F-araAMP when both of thesecomponents are inoculated directly into a tumor mass is investigated(FIG. 5A). In the experiment shown, 2×10¹¹ VP of Ad/PNP are administeredIT twice per day for 3 days (days 15, 16, and 17) for a total of 6injections. The tumors are subsequently inoculated with 24 mg ofF-araAMP once per day on Days 20, 21, and 22. E. coli PNP activity inthe tumors on day 20 (the first day of F-araAMP treatment) is7,500±2,000 units, which is similar to that observed for tumors in which5 to 10% of the cells stably expressed the enzyme (2,500, 14,000, 8,600,or 12,000 units) and within the range for which robust antitumoractivity attributable to IT F-araAMP is expected. Although 2 of 10 micetreated with Ad/PNP plus F-araAMP died, there is an excellent antitumoreffect in the 8 surviving mice who experienced 17% weight loss duringtreatment that later resolved. Because of the evidence of toxicity notedin the experiment shown in FIG. 5A, the study is repeated at a reduceddose of F-araAMP (18 mg/injection). With this schedule, there are nodrug related deaths in the treatment group, and although murine bodyweights did not decrease, prolonged antitumor activity is noted throughday 71 post implant (FIG. 5B). The E. coli PNP activity in the tumors ofthis experiment is 1,900±1,500 units. The results establish thatintratumoral injection of Ad/PNP followed by IT F-araAMP can elicit asubstantial regressive effect on otherwise refractory solid tumors thatis superior to that seen after intratumoral injection of Ad/PNP followedby IP F-araAMP (Hong et al. Cancer Res. 2004; 64: 6610-6615).

Injection of tumors with both Ad/PNP plus higher doses of F-araAMP had adramatic effect on tumor growth (FIGS. 5A and 5B), which is superior toAd/PNP plus systemic F-araAMP.

Example 8 Effect of F-dAdo Versus F-araAMP

F-dAdo or F-araAMP is injected 3 times into D54 tumors in 150 μl volumesin 8 separate injections of approximately 20 μl each. In FIG. 5A,parental D54 human glioma tumors are treated IT with vehicle (closedcircles), Ad/PNP (open circles), F-araAMP (filled triangles), or Ad/PNPplus F-araAMP (open triangles). Ad/PNP (2×10¹¹ VP) is injected twice aday for 3 consecutive days starting on day 15. Twenty four mg F-araAMPdissolved in DMSO is injected into tumors once per day for 3 consecutivedays starting on day 20. The tumors in vehicle treated mice are injected6 times with saline followed by 3 injections of DMSO (otherwise asdescribed above). The activity of E. coli PNP in the D54 tumors (on day20) is 7,500±2,000 PNP units. Each treatment arm contained 10 mice. Inthe combined treatment arm, two of ten mice died as a consequence oftherapy. Four of the eight surviving mice are tumor-free on day 65, twomice had small tumors (63 and 288 mg), and two mice had growing tumors(1666 and 1584 mg). The tumor growth in mice treated with Ad/PNP plusF-araAMP is significantly different than that in mice treated withF-araAMP or Ad/PNP only (P=0.010 and 0.002, respectively). In FIG. 5B,mice are treated as described in FIG. 5A except that the dose ofF-araAMP is 18 mg per injection. The activity of E. coli PNP on day 18in this experiment is 1900±1500 PNP units. With regard to thecombination of Ad/PNP with F-araAMP, on day 71, two mice had very smalltumors (72 and 108 mg), two mice had larger tumors, and two mice died byday 71 due to unknown causes temporally unrelated to F-araAMP toxicity(days 13 and 43). The tumor growth in mice treated with Ad/PNP plusF-araAMP is significantly different than that in mice treated withF-araAMP or Ad/PNP only (P=0.001 and 0.011, respectively). Data using analternative source of T. vaganalis (Tv)- PNP is shown ins FIG. 5C.

Better results are observed with F-araAMP than with F-dAdo (FIGS. 6A and6B) although F-dAdo is a much better substrate for E. coli PNP than isF-araA when the cells express PNP.

Example 9 Effect of Intratumoral F-araAMP and Ad/PNP on DU145 (HumanProstrate) Tumors and NCI-H322M (Human Non-Small Cell AdenocarcinomaLung) Tumors

Injections of F-araAMP and Ad/PNP are performed as detailed above, withthe exception that the tumors are now DU145 with similar results. (FIG.7A). Injections of F-araAMP and Ad/PNP are performed as detailed above,with the exception that the tumors are now H322M with similar results.(FIG. 7B).

Example 10 Effect of Intratumoral Injection of F-araAMP Plus Radiationon D54 Tumors

Locally advanced solid tumors often become resistant to radiationtherapy. As a test of adjuvant E. coli PNP in this setting, F-araAMPinjection together with external beam radiation is investigated. InFIGS. 8A and 8B, tumors in which 10% of cells expressed E. coli PNP areadministered radiation (determined previously to confer a measurableeffect on D54 tumor growth) with or without three IT injections of 3 mgof F-araAMP (a dose that resulted in tumor suppression but no tumorfree-survivors, FIG. 2A). Combining E. coli PNP/F-araAMP with radiationtherapy resulted in a pronounced antitumor activity, which is muchgreater than either treatment alone. Ten to fourteen percent weight lossis observed in all treatment groups (which rapidly resolved aftertermination of treatment), indicating that toxicities of the twointerventions are not additive. Similar results are obtained with thesustained release F-araAMP of Example 5.

D54 tumor cells in which 10% of cells expressed E. coli PNP are injectedsc into the flanks of nude mice. The tumors are treated with radiation,F-araAMP, or F-araAMP plus radiation. In two treatment groups, tumorsare injected once per day for 3 consecutive days with 150 μl of eithersaline (filled circles) or 3 mg F-araAMP dissolved in saline (opencircles). In two other treatment groups radiation (4 Gy) is administeredonce per day for 3 consecutive days 3 hours after injection of 150 μl ofeither saline (filled squares) or 3 mg F-araAMP dissolved in saline(open squares). The activity of E. coli PNP in the D54 tumors (on day16) is 12,000±3,000 units. The tumor growth in mice treated withradiation plus F-araAMP is significantly different than that in micetreated with F-araAMP or radiation only (P=0.004 and <0.001,respectively).

Example 11

Radioactivity in D54 Tumors After Intratumoral Injection of F-araAMP

In an effort to understand the pharmacodynamics of intratumoral (IT)F-araAMP, the levels of prodrug activation in parental (D54) tumors andtumors in which 10% of cells expressed E. coli PNP are monitored (Table1). Tumor tissue is collected 10 minutes or 4 hours after IT injectionof 3 mg [³H]-F-araAMP and the amount of radioactivity remaining in thetumor mass determined. Ten minutes after injection with F-araAMP, thereare no differences between parental and D54 tumors in which 10% of cellsexpress E. coli PNP. By 4 hours, radioactivity in D54 tumors thatexpressed E. coli PNP is substantially higher than parental tumors,representing the amount of F-araAMP converted to F-Ade metabolites. Theexperiment indicated that 190 nmoles of F-Ade metabolites are generatedand retained per gram of tumor tissue after intratumoral injection of 3mg F-araAMP. Three mg of F-araAMP is equal to 8200 nmoles, and sincetumors in this experiment are approximately 0.3 grams, approximately 57nmoles of F-Ade are retained in the tumor tissue in this experiment, or0.7% of the total F-araAMP injected into the tumor.

TABLE 1 Radioactivity in D54 tumors after intratumoral injection ofF-araAMP nmoles F-araAMP/gram of tissue 10 min 4 hours D54 tumors  980 ±730 31 ± 6  10% PNP D54 tumors 1100 ± 990 220 ± 75* Three mg (8,200nmoles) of [³H]F-araAMP (10 μCi/injection) are injected into D54 tumorsor D54 tumors in which 10% of the cells express E. coli PNP as describedin FIG. 2. Tumors (approximately 300 mg) are removed 10 minutes and 4hours after injection with F-araAMP and the amount of radioactivity ineach tumor determined. There are 4 tumors per group. This experiment hasbeen repeated with similar results. *Significantly different from D54tumors: p < 0.02, paired Student's t-test.

Patent documents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. These documents and publications are incorporatedherein by reference to the same extent as if each individual document orpublication was specifically and individually incorporated herein byreference.

The foregoing description is illustrative of particular embodiments ofthe invention, but is not meant to be a limitation upon the practicethereof. The following claims, including all equivalents thereof, areintended to define the scope of the invention.

1. A process to sequentially destroy large regions of a solid tumorcomprising: delivering a purine nucleoside phosphorylase or nucleosidehydrolase or a vector encoding expression thereof directly into thesolid tumor; and injecting intratumorally a prodrug cleaved by saidpurine nucleoside phosphorylase or nucleoside hydrolase directly intothe solid tumor to release a purine base cytotoxic into a portion of thesolid tumor to sequentially destroy large regions of the solid tumor. 2.The process of claim 1 wherein said purine nucleoside phosphorylase ornucleoside hydrolase is delivered with a viral vector containing anucleic acid encoding said purine nucleoside phosphorylase.
 3. Theprocess of claim 2 wherein said viral vector is an adenoviral vector. 4.The process of claim 1 wherein said purine nucleoside phosphorylase ispresent and is a mutant of E. coli.
 5. The process of claim 1 whereinsaid purine nucleoside phosphorylase is present and is a tailed mutant.6. The process of claim 1 further comprising exposing the targeted cellsto X-ray radiation.
 7. The process of claim 1 wherein said purinenucleoside phosphorylase is present and said prodrug is fludarabinephosphate.
 8. The process of claim 1 further comprising a sustainedrelease carrier of a gel, paste, or a microparticle.
 9. The process ofclaim 1 wherein said purine base is a purine base of 2-fluoroadenine.10. The process of claim 1 wherein said prodrug is injected directlyinto proximity to the solid tumor in multiple doses.
 11. The process ofclaim 1 wherein said prodrug is injected directly into proximity to thetargeted cells in at least three consecutive doses.
 12. A process ofperforming a tissue ablation procedure without granulation orscarification comprising: delivering a purine nucleoside phosphorylaseor nucleoside hydrolase or a vector encoding expression thereof directlyinto proximity to the tissue; and injecting intratumorally a prodrugcleaved by said purine nucleoside phosphorylase or nucleoside hydrolasedirectly into proximity to the tissue to release a purine base cytotoxicto the tissue to induce chemical ablation.
 13. The procedure of claim 12wherein the procedure is treatment of one of: cardiac arrhythmia, cyst,reduction, ganglion treatment, male sterilization, cosmeticdermatological procedures, or melanoma.